The need for novel therapeutic and prophylactic approaches to Clostridium difficile (C. difficile) infections in the VA Health Care system, is urgent. The overall goal of this proposal is to develop live oral vaccines which express C. difficile toxin antigens which will induce protective mucosal, and systemic, immunity to the toxins in order to prevent Clostridium difficile-associated disease (CDAD). To achieve this, an attenuated strain of E.coli which we have developed as a vaccine vector for antigen delivery will be engineered to express the immunogenic C-terminal receptor binding portions of the major virulence determinants of C.difficile, the A and B toxins (TcdA and TcdB). To evaluate vaccine efficacy we will use a recently described antibiotic (cefoperazone)-treated mouse model of CDAD(70) which we have validated in our laboratory. To express protective toxin antigens in immunogenic form in our E.coli vector strain, we will incorporate them into the passenger domain of autotransporter(28) proteins to deliver them to the mucosal immune system as we have done for other toxin antigens. This proposal has 3 specific aims: The first specific aim is to express immunogenic components of the TcdA and TcdB toxins of C. difficile as antigens in our attenuated E. coli vector ZCR533(6). The C-terminal binding regions of the toxins will be expressed as fusions with the passenger domain of the autotransporter EspP, the intrinsic type V secretion system(28) of O157:H7 E.coli, and introduced on a medium copy number plasmid. The second specific aim is to determine the safety and mucosal immunogenicity of the vaccine constructs by intranasal, and intragastric, immunization of mice, followed by measurement of the binding and toxin neutralizing capacity ofserum and mucosal anti-TcdA and/or anti-TcdB antibodies. The third specific aim is to determine the protective efficacy of the vaccine constructs in C57/Bl6 mice using a newly described antibiotic-treated mouse model(70) for C. difficile challenge. We have recently validated this model in our laboratory. Our vaccine vector will be ZCR533, an attenuated E.coli strain derived from an O157:H7 enterohemorrhagic E.coli (EHEC) by deleting its genes for shiga toxin production and truncating its major adhesin, intimin, such that it can no longer induce attaching /effacing lesions, but can still induce anti-intimin antibody(8,75). We will use standard molecular genetics techniques to prepare C. difficile vaccine constructs by expressing the C terminal repeats comprising the binding domains of the TcdA and TcdB toxins, into the EspP autotransporter(28) on a multicopy plasmid. Our recent studies have indicated the validity of this approach to immunization against toxin antigens, since the similarly-sized shiga toxin B subunit antigen is immunogenic, and protective against shiga toxin producing E.coli (STEC) infection, when expressed in the autotransporter EspP on the surface of the organism, or secreted into the medium. To test the safety, immunogenicity and protective efficacy of the vaccine constructs we will utilize mouse models of intranasal, and intragastric, immunization(12) and a new cefoperazone-treated mouse model (70) of CDAD, which we have validated in our laboratory, for C. difficille challenge. These studies could lead to the development of a new class of safe and effective vaccines directed against C.difficile toxins to prevent CDAD.